Two pathways regulate insulin-like growth factor genes in the brain and liver of the tropical damselfish Chrysiptera cyanea: A possible role for melatonin in the actions of growth and thyroid hormones.

External and internal factors are involved in controlling the growth of fishes. However, little is known about the mechanisms by which external factors trigger stimulus signals. This study explored the physiological roles of melatonin in the transcription of growth-related genes in the brain and liver of Chrysiptera cyanea, a tropical damselfish with long-day preference. In brain samples of this species collected at 4-h intervals, the transcript levels of arylalkylamine N-acetyltransferase2 (aanat2), the rate-limiting enzyme of melatonin synthesis, and growth hormone (gh) peaked at 20:00 and 00:00, respectively. Concomitantly, the transcript levels of insulin-like growth factors (igf1 and igf2) in the brain and liver were upregulated during the scotophase. Levels of iodothyronine deiodinases (dio2 and dio3), enzymes that convert thyroxine (T4) to triiodothyronine (T3) and reverse T3, respectively, increased in the brain (dio2 and dio3) and liver (dio2) during the photophase, whereas dio3 levels in the liver showed the opposite trend. Fish reared in melatonin-containing water exhibited significant increases in the transcription levels of gh and igf1 in the brain and igf1 in the liver, suggesting that growth in this fish is positively regulated by the GH/IGF pathway on a daily basis. Melatonin treatment also stimulated the transcript levels of dio2 and dio3 in the liver, but not in the brain. Fish consuming pellets containing T3, but not T4, showed significant increases in gh and igf1 in the brain and igf1 and igf2 in the liver, suggesting that the intercellular actions of the TH/IGF pathway have an impact on growth on a daily basis. In summary, IGF synthesis and action in the brain and liver undergo dual regulation by distinct hormone networks, which may also be affected by daily, seasonal, or nutritional factors.

Effects of dopamine and melatonin treatment on the expression of the genes associated with artificially induced sexual maturation in Japanese eel, Anguilla japonica.

Japanese eel (Anguilla japonica) is a commercially important fish species in Asia. Understanding factors like photoperiod, temperature, and lunar cycles is crucial for successful aquaculture and managing its reproduction. Melatonin and dopamine (DA) are essential for regulating reproduction in vertebrates, including fish. This study investigated the effects of melatonin and DA on the reproductive system of mature male Japanese eels to better understand reproductive regulation in fish. To clarify the effects of these hormones on sexual maturation in eels, a critical stage in the reproductive process, sexual maturation was induced by injecting human chorionic gonadotropin, which stimulates the production of sex hormones. To check the effect of melatonin and DA on sexual maturation, DA, melatonin, and DA + domperidone were intraperitoneally injected into fish from each group (six per treatment) at a dose of 1 mg/kg body weight. The fish were then examined using quantitative RT-PCR by comparing the messenger RNA level of reproduction-related genes (gonadotropin releasing hormone 1; gnrh1, gonadotropin releasing hormone 2; gnrh2, follicle stimulating hormone; fshß, luteinizing hormone; lhß and DA receptor 2b; d2b), involved in the gonadotropic axis in eels, to those that received a control injection. The results indicate significant differences in the expression levels of gnrh1, gnrh2 and d2b in the brain and d2b, fshß, lhß in the pituitary at different stages of sexual maturation. Melatonin appears to enhance the production of sex gonadotropins, whereas DA inhibits them. These findings suggest an interaction between melatonin and DA in regulating reproduction in Japanese eels.

DNA barcoding and morphological identification of spiny lobsters in South Korean waters: a new record of Panulirus longipes and Panulirus homarus homarus.

To date, 19 species of spiny lobsters from the genus Panulirus have been discovered, of which only P. japonicus, P. penicilatus, P. stimpsoni, and P. versicolor have been documented in South Korean waters. In this study, we aimed to identify and update the current list of spiny lobster species that inhabit South Korean waters based on the morphological features and the phylogenetic profile of cytochrome oxidase I (COI) of mitochondrial DNA (mtDNA). Spiny lobsters were collected from the southern and eastern coasts of Jeju Island, South Korea. Phylogenetic analyses were performed using neighbor-joining (NJ), maximum likelihood (ML), and Bayesian inference (BI) methods. The ML tree was used to determine the spiny lobster lineages, thereby clustering the 17 specimens collected in this study into clades A, B, C, and D, which were reciprocally monophyletic with P. japonicus, P. homarus homarus, P. longipes, and P. stimpsoni, respectively. These clades were also supported by morphological examinations. Interestingly, morphological variations, including the connected pleural and transverse groove at the third abdominal somite, were observed in four specimens that were genetically confirmed as P. japonicus. This finding is novel within the P. japonicus taxonomical reports. Additionally, this study updates the documentation of spiny lobsters inhabiting South Korean waters as P. longipes and P. homarus homarus were recorded for the first time in this region.

Testis development in the Japanese eel is affected by photic signals through melatonin secretion.

OBJECTIVE: According to reported spawning characteristics of Japanese eel, Anguilla japonica, which exhibit spawning and migration patterns that are synchronized with lunar cycles and photoperiod, we hypothesized that a close association exists between specific photic signals (daylight, daylength, and moonlight) and endocrinological regulation. Given the photic control in melatonin secretion, this hypothesis was tested by investigating whether melatonin signals act as mediators relaying photic signals during testis development in the eel. METHODS: We examined changes in melatonin-secretion patterns using time-resolved fluorescence immunoassays in sexually immature and mature male Japanese eels under the condition of a new moon (NM) and a full moon (FM). RESULTS: The eye and plasma melatonin levels exhibited a nocturnal pattern under a 12-h light: dark cycle (12L12D) or under constant darkness (DD), but not with constant light (LL). Eye melatonin levels were similar under the 12L12D and short-day (9L15D) conditions. In the long-day condition (15L9D), secreted plasma melatonin levels were stable, whereas short-day melatonin secretion began when darkness commenced. Sexual maturation began at 8 weeks following intraperitoneal injection of human chorionic gonadotropin (hCG), and NM exposure led to significantly higher eye and plasma melatonin levels compared with those detected under FM exposure.

Food availability alters expression profiles of genes in relation to reproduction and nutrition in the females of tropical damselfish (Chrysiptera cyanea).

This study evaluated the effects of food availability on the transcript levels of genes related to reproduction and growth in the sapphire devil (Chrysiptera cyanea), a tropical damselfish. Nonbreeding fish were reared at high-food (HF) and low-food (LF) levels for 4 weeks under long-days. Vitellogenic oocytes could be observed in the ovaries of the HF group. The quantitative polymerase chain reaction analysis revealed that lhß and cyp19b in the brains, vtg and igf1 in the livers and cyp19a in the ovaries of HF fish were significantly higher than that of LF fish, suggesting that estradiol-17ß (E2) synthesis in the ovary and brain is activated when suitable permissive factors are available to fish. Food limitation lowered hepatic igf1 and dio2, suggesting that the TH-IGF1 signaling system functions in the liver, and that food availability altered hepatic deiodination activities related to intercellular levels of thyroid hormones. Hepatic dio2 significantly decreased when fish were immersed for 3 days in E2-containing seawater; this suggests that E2 impedes the conversion of T4 to T3 in the liver. Our study shows that igf1 was upregulated in accordance with HF-induced vitellogenesis but downregulated by E2 treatment, suggesting that igf1 is bidirectional and altered by maturational status. Once vitellogenesis begins under a suitable range of proximal factors, fish need to maintain their nutritional status because food availability is a permissive factor for their reproduction.

Verification of differentially expressed genes in relation to hydrostatic pressure in the brain of two wrasse species with high-tide preference in spawning.

Fish that inhabit shallow water are exposed to periodic changes in tidal cues, including hydrostatic pressure (HP). The present study aimed at verifying differentially expressed genes (DEGs) in the brain of the threespot wrasse Halichoeres trimaculatus (tropical species) and the honbera wrasse Halichoeres tenuispinis (temperate species), both of which were exposed to HP at 30 kPa (possible high-tide stimuli in the field) or 1 kPa (low tide) for 3 or 6 h. A de novo assembly yielded 174,710 contigs (63,530 contigs were annotated) from the brain of threespot wrasse. Following RNA sequencing, quantitative PCR confirmed DEGs that were upregulated [AT atypical cadherin 2 (FAT2)] and downregulated [neuronal leucine-rich repeat protein 3 (LRRN3), dual specificity tyrosine phosphorylation-regulated kinase 1 (DYRK), mitogen-activated protein kinase kinase 1 (MAP2K1) and phosphoinositide 3 kinase (PI3K)]. The effect of HP on the transcription of these DEGs (except for MAP2K1) disappeared within 6 h, suggesting that HP is a transitory stimulus occurring at the beginning of the tidal cycle. Similar DEG transcription was observed in the brain of honbera wrasse maintained under HP for 6 h. In situ hybridization of the brain of the threespot wrasse revealed that strong signals of MPA2K1 were seen in the telencephalon, diencephalon and pituitary, whereas those of PI3K were seen in the telencephalon, diencephalon and medulla oblongata. This result suggests that these kinases are involved in sensory function (telencephalon), somatic and visceral function (medullar oblongata) and the neuroendocrine system (diencephalon and pituitary), all of which were related to changes in HP stimuli. Following HP exposure, the transcription of c-fos increased in the pituitary of honbera wrasse, suggesting that external stimuli directly or indirectly activate hormone synthesis at the hypothalamic-pituitary-gonadal axis. It is concluded that HP alters gene expression in relation to neural development and function in the central nervous system and plays a role in exerting tidal-related reproduction and feeding in wrasses.

Molecular cloning of insulin-like growth factor 3 (igf3) and its expression in the tissues of a female damselfish, Chrysiptera cyanea, in relation to seasonal and food-manipulated reproduction.

Food availability is a permissive determinant that drives gonadal activity in fish. The present study aimed to clarify the interactions between reproductive and nutritive statuses in the sapphire devil (Chrysiptera cyanea), a tropical damselfish with a long-day preference for reproduction. Insulin-like growth factor 3 (IGF3), a novel IGF that likely plays a role in gonadal maturation, was closely monitored in the sapphire devil. The cDNA of sapphire devil igf3 had an open reading frame of 443 base pairs (146 amino acid residues). Phylogenetic analyses revealed that sapphire devil IGF3 was clustered within the teleost IGF3 family. The transcript levels of sapphire devil igf3 increased in the brain, liver, and ovary of the fish during the late vitellogenic phase, suggesting that it plays a role in reproduction. Immersion of the fish in seawater containing estradiol-17ß suppressed transcript levels of sapphire devil igf3 in the liver, but not in the brain, suggesting that intensive protein synthesis in relation to vitellogenesis negatively impacts somatic metabolism in this tissue. When fish were reared with high or low food under conditions of photoperiod (LD = 14:10) and temperature (at 25-28 °C) during the non-reproductive season, ovarian development was induced in high-food fish. Furthermore, prior to ovarian development in the high-food fish, the transcript levels of sapphire devil igf3 increased in the brain, liver, and ovary. These results indicated crosstalk between the reproductive and growth networks and suggested that a metabolic shift, from growth mode to reproductive mode, occurs in peripheral tissues when nutritive status is improved under suitable conditions of photoperiod and water temperature.

Gene expression patterns of novel visual and non-visual opsin families in immature and mature Japanese eel males.

This study was carried out to identify and estimate physiological function of a new type of opsin subfamily present in the retina and whole brain tissues of Japanese eel using RNA-Seq transcriptome method. A total of 18 opsin subfamilies were identified through RNA-seq. The visual opsin family included Rh2, SWS2, FWO, DSO, and Exo-Rhod. The non-visual opsin family included four types of melanopsin subfamily (Opn4x1, Opn4x2, Opn4m1, and Opn4m2), peropsin, two types of neuropsin subfamily (Opn5-like, Opn5), Opn3, three types of TMT opsin subfamily (TMT1, 2, 3), VA-opsin, and parapinopsin. In terms of changes in photoreceptor gene expression in the retina of sexually mature and immature male eels, DSO mRNA increased in the maturation group. Analysis of expression of opsin family gene in male eel brain before and after maturation revealed that DSO and SWS2 expression in terms of visual opsin mRNA increased in the sexually mature group. In terms of non-visual opsin mRNA, parapinopsin mRNA increased whereas that of TMT2 decreased in the fore-brain of the sexually mature group. The mRNA for parapinopsin increased in the mid-brain of the sexually mature group, whereas those of TMT1 and TMT3 increased in the hind-brain of the sexually mature group. DSO mRNA also increased in the retina after sexual maturation, and DSO and SWS2 mRNA increased in whole brain part, suggesting that DSO and SWS2 are closely related to sexual maturation.

Daily variation of D2 dopamine receptor transcription in the brain of the Japanese eel Anguilla japonica and its regulation with dopamine and melatonin.

Dopamine plays a crucial role in controlling reproduction in eels, and its action is mediated through D2-type dopamine receptors. D2A and D2B receptors in the Japanese eel Anguilla japonica were cloned and characterized in the present study. Attention (daily expression patterns in the brain and endogenous regulation) was paid to D2B receptor because it is considered to play a crucial role in eel reproduction. The cDNAs of D2A and D2B receptors had open reading frames comprising 456 and 454 amino acid residues, respectively, which were phylogenetically clustered with those of other teleost species. Both receptors were highly expressed in the brain. D2B receptor transcript levels exhibited high day/low night variation in the midbrain and pituitary, suggesting that its transcription in these tissues is regulated in a daily manner, possibly under influence of melatonin. Intraperitoneal injection of dopamine downregulated D2B receptor transcription significantly in the midbrain and moderately in the pituitary within 1 h, but upregulated its transcription in the forebrain. Co-injection of dopamine with its antagonist (domperidone) reversed the effect of dopamine in the pituitary and forebrain, but not in the midbrain, suggesting that the effect of dopamine on D2B receptor transcription differs among brain regions. The same treatment with melatonin resulted in decreased D2B receptor transcription in the midbrain. These findings indicate that dopamine and melatonin have key roles in the daily variation in D2B receptor transcription in the brain of Japanese eel, and that they are related to a daily base secretion of hormones in the hypothalamic-pituitary-gonadal axis in this species.

Daily expression of a clock gene in the brain and pituitary of the Malabar grouper (Epinephelus malabaricus).

Recent studies have revealed that, in addition to regulating the circadian system, clock genes such as cryptochrome (Cry) genes are involved in seasonal and lunar rhythmicity in fish. This study clarified the transcriptional characteristics of a Cry subtype (mgCry2) in the brain of the Malabar grouper, Epinephelus malabaricus, which is an important aquaculture species that spawns around the new moon. The cDNA sequence of mgCry2 showed high identity (97-99%) with fish Cry2 and had an open reading frame encoding a protein with 170 amino acids. Phylogenetic analyses revealed that mgCRY2 had high identity with CRY in other fish species. Real-time quantitative polymerase chain reaction (qPCR) showed the widespread distribution of mgCry2 in neural (brain, pituitary, and retina) and peripheral (heart, liver, kidney, spleen, gill, intestine, and ovary) tissues. When immature Malabar groupers were reared under a light-dark cycle (LD = 12:12) and the amounts of mgCry2 mRNA in the telencephalon and diencephalon were measured at 4-h intervals, the levels increased during photophase and decreased during scotophase. Day-night variation in mgCry2 mRNA abundance was also observed in the pituitary. These daily profiles suggest that mgCry2 is a light-responsive gene in neural tissues. In situ hybridization analyses showed that mgCry2 was strongly transcribed in the nucleus lateralis tuberis of the ventral hypothalamus, peripheral area of the proximal pars distalis, and the pars intermedia of the pituitary. We conclude that clock genes expressed in the pituitary and diencephalon play a role in entraining the endocrine network of the Malabar grouper to periodic changes in external cues.

Involvement of Estrogen and Its Receptors in Morphological Changes in the Eyes of the Japanese Eel, Anguilla japonica, in the Process of Artificially-Induced Maturation.

During the long migration from river habitats to the spawning ground, the Japanese eel undergoes sexual maturation. This spawning migration occurs concurrently with morphological changes, such as increases in eye size; however, the mechanisms by which sex steroids and their receptors influence these changes in peripheral tissues remain unclear. The aim of this study was to investigate changes in the eyes of female Japanese eels during sexual maturation, and our research focused on estrogen receptor (ER)α and ERß transcripts. During ovarian development, the gonadosomatic index increased and yolk-laden oocytes developed rapidly. These changes occurred in conjunction with a steady increase in plasma levels of estradiol-17ß (E2). Concomitant increases in transcript levels of ERα and ERß in eye, brain, pituitary, and ovary were also observed. Fluorescence in-situ hybridization analyses revealed that ERα and ERß transcripts were present in the choriocapillary layer and photoreceptor layer of the eyes, and the analysis also revealed that their signals in these layers became stronger in mature females compared to those observed in immature females, suggesting that under the influence of gonadotropins, morphological changes in the eyes are regulated by E2 through the activation of its receptors. In conclusion, E2 plays a crucial role in physiological adaptations that occur in peripheral tissues during the spawning migration.